Varna Research Articles

Complementation of adenovirus virus-associated RNA I gene deletion by expression of a mutant eukaryotic translation initiation factor.

Adenovirus VA RNAs (virus-associated RNAs) are small polymerase III transcripts that are required for efficient initiation of mRNA translation late in adenovirus infection. VAI RNA prevents double-stranded RNA (dsRNA) activation of the interferon-induced protein kinase (DAI kinase). Activation of this kinase results in phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF-2 alpha) and correlates with inhibition of translation initiation. In this report we show growth complementation of adenoviruses harboring deletions in the VAI gene in cell lines expressing a serine-to-alanine mutant of eIF-2 alpha. This serine-to-alanine mutant is resistant to phosphorylation by DAI kinase. These results directly show that the primary function of VAI RNA in the lytic adenovirus infection is the inhibition of eIF-2 alpha phosphorylation by DAI kinase and identify eIF-2 alpha as the target that mediates the effects of DAI kinase activation. Cells that express a mutant eIF-2 alpha will enable the isolation of specific host-range mutants for other types of viruses that are defective in the ability to inhibit DAI kinase.

Cis-acting elements and a trans-acting factor affecting alternative splicing of adenovirus L1 transcripts.

Expression of the L1 region of adenovirus is temporally regulated by alternative splicing to yield two major RNAs encoding the 52- to 55-kilodalton (52-55K) and IIIa polypeptides. The distal acceptor site (IIIa) is utilized only during the late phase of infection, whereas the proximal site (52-55K) is used at both early and late times. Several parameters that might affect this alternative splicing were tested by using expression vectors carrying the L1 region or mutated versions of it. In the absence of a virus-encoded or -induced factor(s), only the 52-55K acceptor was used. Decreasing the distance between the donor and the IIIa acceptor had no effect. Removal of the 52-55K acceptor induced IIIa splicing slightly, implying competition between the two acceptors. Fusion of the IIIa exon to the 52-55K intron greatly enhanced splicing of the IIIa junction, suggesting that the IIIa exon does not contain sequences that inhibit splicing. Thus, the lack of splicing to the IIIa acceptor in the absence of a virus-encoded or -induced factor(s) is probably due to the absence of a favorable sequence and/or the presence of a negative element 5' of the IIIa splice junction, or both. The presence of several adenovirus gene products, including VA RNAs, the E2A DNA-binding protein, and the products of E1A and E1B genes, did not facilitate use of the IIIa acceptor. In contrast, the simian virus 40 early proteins, probably large T antigen, induced IIIa splicing. This result, together with those of earlier studies, suggest that T antigen plays a role in modulation of alternative RNA splicing.

Cis-Acting Elements and a trans-Acting Factor Affecting Alternative Splicing of Adenovirus L1 Transcripts

Expression of the L1 region of adenovirus is temporally regulated by alternative splicing to yield two major RNAs encoding the 52- to 55-kilodalton (52-55K) and IIIa polypeptides. The distal acceptor site (IIIa) is utilized only during the late phase of infection, whereas the proximal site (52-55K) is used at both early and late times. Several parameters that might affect this alternative splicing were tested by using expression vectors carrying the L1 region or mutated versions of it. In the absence of a virus-encoded or -induced factor(s), only the 52-55K acceptor was used. Decreasing the distance between the donor and the IIIa acceptor had no effect. Removal of the 52-55K acceptor induced IIIa splicing slightly, implying competition between the two acceptors. Fusion of the IIIa exon to the 52-55K intron greatly enhanced splicing of the IIIa junction, suggesting that the IIIa exon does not contain sequences that inhibit splicing. Thus, the lack of splicing to the IIIa acceptor in the absence of a virus-encoded or -induced factor(s) is probably due to the absence of a favorable sequence and/or the presence of a negative element 5' of the IIIa splice junction, or both. The presence of several adenovirus gene products, including VA RNAs, the E2A DNA-binding protein, and the products of E1A and E1B genes, did not facilitate use of the IIIa acceptor. In contrast, the simian virus 40 early proteins, probably large T antigen, induced IIIa splicing. This result, together with those of earlier studies, suggest that T antigen plays a role in modulation of alternative RNA splicing.

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Varna qui signifie couleur dans le sens de caracteristique ou, mieux, de classe , constitue le principal element taxonomique des textes vediques. Il definit un reseau de hierarchies verticales et horizontales dans les mythes cosmogoniques et legitime ainsi un ordre social fonde sur un systeme de castes

On polynomials with curved majorants

At a conference held in Varna, Bulgaria in the year 1970 the late Professor P. Turan proposed the following problem

Modification of protein synthesis initiation factors and the shut-off of host protein synthesis in adenovirus-infected cells

A substantial body of data, largely derived from study of cell extracts, indicates that protein synthesis in adenovirus-infected cells requires VA RNA 1 at late times of infection to prevent the activation of a protein kinase known as DAI, and the consequent phosphorylation of the a-subunit of initiation factor eIF-2. To verify this conclusion, we have measured the steady-state levels of eIF-2α phosphorylation in cells infected with wild-type virus (Ad2) and a mutant that produces no VA RNA, (Ad5 dl331). Consistent with the proposed mechanism, the a-subunit was very highly phosphorylated (∼90%) at late times of infection with Ad5 dl331. Surprisingly, eIF-2α phosphorylation also increased (to ∼30%) at late times of infection with Ad2, suggesting that VA RNA and DAI might be involved in the selective translation of viral mRNA and the shut-off of host cell protein synthesis during the late phase. In agreement with this model, host protein synthesis shut-off is defective in cells expressing low levels of DAI.

Purification of transcription factor IIIB from HeLa cells.

Transcription factor IIIB (TFIIIB), which by itself does not bind stably or specifically to DNA, was purified from cytoplasmic extracts of HeLa cells using five different chromatographic steps. This procedure yields one predominant polypeptide which represents 90% of the most highly purified preparation and shows a relative molecular mass of 60,000, when analyzed on sodium dodecyl sulfate-polyacrylamide gels. A similar value was obtained for the native protein by rate zonal centrifugation on glycerol gradients. From these data we conclude that TFIIIB from HeLa cells has a Mr of 60,000 +/- 5,000 and that it functions as a single polypeptide. Highly purified TFIIIB was required and sufficient for the specific transcription of the Xenopus laevis and human tRNA and 5 S RNA genes as well as those for VA RNA when reconstituted with RNA polymerase III and the other appropriate transcription factors.

Characterization of the double-stranded RNA implicated in the inhibition of protein synthesis in cells infected with a mutant adenovirus defective for VA RNA

In the absence of VA RNA,, protein synthesis in adenovirus-infected HeLa cells fails at late times of infection because of defective initiation. The defect is due to the activation of a protein kinase that phosphorylates the α-subunit of initiation factor eIF-2. The kinase responsible for the translational defect is DAI, the double-stranded RNA (dsRNA)-activated inhibitor of protein synthesis, which is present in uninfected HeLa cells at a basal level and in a largely inactive, latent state. In vitro it can be activated by incubation with ATP and a low concentration of dsRNA. Previous studies suggested that RNA generated during the course of infection can activate DAL We show here that the activator RNA has the properties of dsRNA: it chromatographs with dsRNA, can be denatured and reannealed, and is destroyed by a dsRNA-specific nuclease. At least some of the dsRNA is viral. It hybridizes to DNA sequences in the center of the viral genome, principally between map units 47 and 51 and 73 and 76, consistent with an origin in the symmetrical transcription of both viral DNA strands.

Cloning of the Human Thyrotropin β-Subunit Gene and Transient Expression of Biologically Active Human Thyrotropin after Gene Transfection

A 17 kilobase pair fragment of DNA containing the human TSH (hTSH) beta-subunit gene was isolated from a human leukocyte genomic library. Using a 621 base pair human CG alpha-subunit cDNA and a 2.0 kilobase pair genomic fragment of hTSH beta containing both coding exons, we constructed hCG alpha and hTSH beta expression vectors containing either the early promoter of simian virus 40 or the promoters of adeno-associated virus. Cotransfection of two adeno-associated virus vectors, each containing one subunit of hTSH, together with a plasmid containing the adenovirus VA RNA genes produced hTSH as well as free human alpha- and TSH beta-subunits in an adenovirus transformed human embryonal kidney cell line (293). The levels of protein expression in this system were 10- to 100-fold greater than that found in a simian virus transformed monkey kidney cell line (COS) using vectors containing the early promoter of simian virus 40. The hTSH synthesized in 293 cells was glycosylated as indicated by complete binding to concanavalin A-Sepharose but was larger in apparent molecular weight than a standard hTSH preparation on gel chromatography suggesting an altered glycosylation pattern. However, it was immunologically and biologically indistinguishable from two pituitary hTSH standards in an immunoradiometric and in vitro iodide trapping assay, respectively.

Multiple proteins bind to VA RNA genes of adenovirus type 2.

Using fractionated HeLa cell nuclear extracts and both nuclease (DNase I) cleavage and chemical cleavage (methidiumpropyl-EDTA X Fe(II) protection methodologies, we demonstrated the presence of three proteins which interacted specifically, yet differentially, with the two VA genes of adenovirus type 2. One, previously identified as transcription initiation factor TFIIIC, bound to a site centered on the transcriptionally essential B-block concensus element of the VAI gene and, with a lower affinity, to the analogous site in the VAII gene. Another, identified as the cellular protein involved in adenovirus replication, nuclear factor I, bound to sites immediately downstream from the two VAI terminators (at approximately +160 and +200). The third, a previously unrecognized VA gene binding protein termed VBP, bound immediately upstream of the B-block element in the VAI gene but showed no binding to VAII. Possible roles for these proteins in VA gene transcription were investigated in in vitro assay systems reconstituted with partially purified transcription factors (RNA polymerase III, TFIIIB, and TFIIIC). Although TFIIIC activity was present predominantly in fractions containing B-block binding activity, there was not complete correspondence between functional and DNA binding activities. The nuclear factor I-like protein had no effect when added to a complete transcription reaction. The presence of VBP appeared to depress the intrinsic ratio of VAI-VAII synthesis, thereby simulating the relative transcription levels observed early in adenovirus infection of HeLa cells. These observations suggest a model, involving both intragenic binding factors (VBP and TFIIIC) and variable template concentrations, for the differential regulation of VA transcription during the course of adenovirus infection.

Multiple proteins bind to VA RNA genes of adenovirus type 2.

Using fractionated HeLa cell nuclear extracts and both nuclease (DNase I) cleavage and chemical cleavage (methidiumpropyl-EDTA X Fe(II) protection methodologies, we demonstrated the presence of three proteins which interacted specifically, yet differentially, with the two VA genes of adenovirus type 2. One, previously identified as transcription initiation factor TFIIIC, bound to a site centered on the transcriptionally essential B-block concensus element of the VAI gene and, with a lower affinity, to the analogous site in the VAII gene. Another, identified as the cellular protein involved in adenovirus replication, nuclear factor I, bound to sites immediately downstream from the two VAI terminators (at approximately +160 and +200). The third, a previously unrecognized VA gene binding protein termed VBP, bound immediately upstream of the B-block element in the VAI gene but showed no binding to VAII. Possible roles for these proteins in VA gene transcription were investigated in in vitro assay systems reconstituted with partially purified transcription factors (RNA polymerase III, TFIIIB, and TFIIIC). Although TFIIIC activity was present predominantly in fractions containing B-block binding activity, there was not complete correspondence between functional and DNA binding activities. The nuclear factor I-like protein had no effect when added to a complete transcription reaction. The presence of VBP appeared to depress the intrinsic ratio of VAI-VAII synthesis, thereby simulating the relative transcription levels observed early in adenovirus infection of HeLa cells. These observations suggest a model, involving both intragenic binding factors (VBP and TFIIIC) and variable template concentrations, for the differential regulation of VA transcription during the course of adenovirus infection.

Transcription complexes for various class III genes differ in parameters of formation and stability towards salt

RNA polymerase III faithfully transcribes the genes for ribosomal 5 S RNA, tRNA 1 Met or adenovirus VA RNA in vitro in the presence of required transcription factors. These genes display distinct differences in the kinetics of transcription complex formation and in their response to excess template. In contrast to tRNA and VA RNA synthesis, 5 S RNA synthesis displays a lag phase of 15 minutes before the onset of transcription and is clearly inhibited by high concentrations of template. Once formed, transcription complexes for the RNA polymerase III genes listed can be isolated by glycerol gradient centrifugation and display a remarkable stability against transient treatment with high salt concentrations. Complexes for 5 S RNA and tRNA remain functionally active up to 2.5 m-KCl. The activity of transcription complexes for VA RNA, however, is significantly diminished after treatment with high salt concentrations. This effect is shown to be due to an irreversible loss of transcription factors. RNA polymerase III is dissociated by high salt concentrations from all the transcription complexes studied but remains part of these complexes during the normal reinitiation cycle at 60 m m-KCl. An additional method for the purification of partial transcription complexes was developed that involves equilibrium centrifugation on cesium sulfate gradients. This method completely releases TFIIIB from 5 S complexes and a core complex, composed of the 5 S RNA gene, factors IIIA and IIIC, is retained. In the case of tRNA and VA RNA, core complexes are obtained that remain partly associated with TFIIIC and TFIIIB. These results indicate a qualitatively and/or quantitatively different interaction of individual factors in different polymerase III transcription complexes.

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The Varna Convention : A Regional Response to Fisheries Conservation and Management

Novobiocin interferes with the binding of transcription factors TFIIIA and TFIIIC to the promoters of class III genes.

Novobiocin has been shown to inhibit class III gene transcription from both chromatin and non-chromatin templates. Since novobiocin is a well characterized inhibitor of type II DNA topoisomerases, it has been postulated that a gyrase activity is necessary for transcription. Using DNase I footprinting, we show here that novobiocin inhibits the specific binding of polymerase III transcription factors TFIIIA and TFIIIC to the promoters of the 5S RNA and VA RNA genes, respectively. Concentrations of novobiocin employed were comparable to those necessary to inhibit HeLa topoisomerase II. In vitro transcription assays, performed under equivalent conditions, demonstrated that similar novobiocin concentrations were necessary for transcription inhibition. These results strongly suggest that novobiocin interferes with transcription by inhibiting specific protein-DNA interactions.

Separation of TFIIIC into two functional components by sequence specific DNA affinity chromatography.

Recently, it has been shown that mammalian transcription factor IIIC (TFIIIC) activity can be separated by anion exchange FPLC chromatography into two functional components (1), both of which are required for transcription of tRNA and the adenovirus VA RNA genes. Here we show that these two functional components, designated TFIIIC1 and TFIIIC2, can also be separated by sequence specific DNA affinity chromatography. These results confirm the observation that TFIIIC can be fractionated into two components, which are both required for transcription of VA I and tRNA genes in vitro. Thus in the mammalian reconstituted system, a minimum of three proteins, in addition to RNA polymerase III, are required for the transcription of the VA and tRNA genes in vitro. The DNA binding component, TFIIIC2, binds specifically to the 3' segment of the internal promoter (the B block), demonstrated by its ability to protect this region from digestion by DNase I. TFIIIC2 is the limiting, titratable component in the phosphocellulose C fraction required for the formation of a stable pre-initiation complex on the VAI RNA gene in vitro, as demonstrated with a template competition and rescue assay.

Suppression of the translation defect phenotype specific for a virus-associated RNA-deficient adenovirus mutant in monkey cells by simian virus 40.

Human cells infected with adenovirus type 2 (Ad2) or Ad5 require VAI RNA for efficient translation of viral mRNAs at late times after infection. The Ad5 mutant dl-sub720 synthesized neither virus-associated I (VAI) nor VAII RNAs, and infection of human cells with this mutant resulted in reduced virion polypeptide synthesis. Infection of monkey cells with this mutant also resulted in drastic reduction of polypeptide synthesis compared with wild-type (WT) adenovirus infections. Steady-state levels of hexon-specific mRNA were found to be comparable in WT- and mutant-infected monkey cells. The in vitro translation experiments showed that double-mutant- and WT-infected cells contained comparable levels of translatable hexon mRNA (and other adenovirus late mRNAs), suggesting that the severe inhibition of hexon protein synthesis in the VA mutant involves a translation block. Preinfection of monkey cells with simian virus 40 fully restored the efficient translation of this mRNA in the VA mutant infections to the level observed in WT-infected cultures. These results raise the possibility that simian virus 40 may encode or induce factors that suppress the translation block that occurs during adenovirus infections in the absence of the VA RNAs.

Rapid enrichment of HeLa transcription factors IIIB and IIIC by using affinity chromatography based on avidin-biotin interactions.

Plasmid DNA containing the adenovirus type 2 genes for VA RNA was linearized at a site distal to the gene, end labeled with a biotin-nucleotide analog of TTP, and incubated with avidin to form an avidin-biotinylated DNA complex. HeLa cell S100 extracts containing crude RNA polymerase III and transcription factors (TFs) IIIB and IIIC were programmed with the avidin-biotin-VA DNA to allow stable complex formation (A.B. Lassar, P.L. Martin, and R.G. Roeder, Science 222:740-748, 1983). Chromatography of the programmed extract over a biotin-cellulose affinity resin resulted in the selective, and virtually quantitative, retention of one of two stable preinitiation complexes, either VA-IIIC or VA-IIIC-IIIB, depending on the length of template incubation in the S100 extract. After washing the resin with 0.10 M and 0.25 M KCl to remove RNA polymerase III and nonspecifically bound proteins, respectively, TFIIIC was eluted from the VA-IIIC complex by the addition of 1.5 M KCl. The VA-IIIC-IIIB complex exhibited a higher salt stability. Most of TFIIIB and some TFIIIC were released by the addition of 1.5 M KCl; however, the majority of TFIIIC activity was recovered only after a subsequent 3.0 M KCl elution. The specific activity of the TFIIIC in the 3.0 M KCl fraction was 770-fold higher than that in the S100 extract, while the protein content of the 1.5 and 3.0 M KCl fractions was reduced 7,500- and 100,000-fold, respectively.

Rapid enrichment of HeLa transcription factors IIIB and IIIC by using affinity chromatography based on avidin-biotin interactions.

Plasmid DNA containing the adenovirus type 2 genes for VA RNA was linearized at a site distal to the gene, end labeled with a biotin-nucleotide analog of TTP, and incubated with avidin to form an avidin-biotinylated DNA complex. HeLa cell S100 extracts containing crude RNA polymerase III and transcription factors (TFs) IIIB and IIIC were programmed with the avidin-biotin-VA DNA to allow stable complex formation (A.B. Lassar, P.L. Martin, and R.G. Roeder, Science 222:740-748, 1983). Chromatography of the programmed extract over a biotin-cellulose affinity resin resulted in the selective, and virtually quantitative, retention of one of two stable preinitiation complexes, either VA-IIIC or VA-IIIC-IIIB, depending on the length of template incubation in the S100 extract. After washing the resin with 0.10 M and 0.25 M KCl to remove RNA polymerase III and nonspecifically bound proteins, respectively, TFIIIC was eluted from the VA-IIIC complex by the addition of 1.5 M KCl. The VA-IIIC-IIIB complex exhibited a higher salt stability. Most of TFIIIB and some TFIIIC were released by the addition of 1.5 M KCl; however, the majority of TFIIIC activity was recovered only after a subsequent 3.0 M KCl elution. The specific activity of the TFIIIC in the 3.0 M KCl fraction was 770-fold higher than that in the S100 extract, while the protein content of the 1.5 and 3.0 M KCl fractions was reduced 7,500- and 100,000-fold, respectively.

A mechanism for the control of protein synthesis by adenovirus VA RNA I

In the absence of VA RNA I, protein synthesis in adenovirus-infected HeLa cells fails because of defective initiation. Earlier work showed that the defect results from phosphorylation of the initiation factor elF-2 on its α subunit. We have identified the protein kinase responsible as the dsRNA-activated inhibitor of protein synthesis (DAI). DAI is present in uninfected HeLa cells at a basal level and in a largely inactive state. It is activated in cells infected with the adenovirus mutant Ad5 dl331, which produces no VA RNA I, but not in cells infected with wild-type virus. Activation occurs during the late phase of infection with the mutant virus, and the activator appears to be dsRNA produced by symmetrical transcription of the viral genome. VA RNA I antagonizes the activation of DAI by dsRNA, but it cannot inhibit the activity of DAI once activated. We propose a mechanism for VA RNA I action based on its partially double-stranded nature.

Anatomy of region L1 from adenovirus type 2

The structure of r-strand-specific RNAs encoded between coordinates 26 and 32 on the adenovirus type 2 genome was mapped by a combination of S1 endonuclease analysis, primer extension, and in vitro transcription. The region includes the third leader segment (coordinates 26.8 to 27.0), the genes for the low-molecular-weight virus-associated RNAs (VA RNAs) (coordinates 29.5 to 30.7), and the amino-terminal end of the gene for the L1 52,000-55,000 polypeptide (coordinates 30.7 to 32.1). The positions at which the tripartite leader was attached to the three longest L1 mRNAs were mapped at the nucleotide level. The leader splice junction of species L1a was located at coordinate 26.8 and coincided with the 3' splice site for the third leader segment, whereas the leader-body splice junction of species L1b and L1c were located at coordinates 29.0 and 30.7, respectively. No protein products have so far been assigned to the L1a and L1b mRNAs, although it can be predicted from the nucleotide sequence that species L1b encodes a 8,300 polypeptide. The third RNA, species L1c, encodes the well-characterized 52,000-55,000 polypeptide. It was also shown that a previously unidentified class of VA RNAs exists predominantly in the poly(A)-fraction of late RNA preparations. These RNAs are heterogeneous in length (up to 3,000 nucleotides) because of irregular transcription termination and have 5' ends which map precisely to the initiation sites for VA RNAI and VA RNAII transcription. Finally it was shown that an RNA with a 5' end coinciding with the 5' splice site for the third leader segment exists in the poly(A)-fraction of late cytoplasmic RNA. This RNA species might represent an excised intron.